1 Final Report: Gasoline Ethanol Vapor Conde nsate: A 13-Week Whole Body Inhalation Toxicity Study in Rats with Neurotoxicity As sessments and a 4-Week In Vivo Genotoxicity and Immunotoxicity Assessmen ts (HLS Study No. 00-6127)

211(b) Research Group (RG) response to EPA Second Draft report Comments Dated April 9, 2008

EPA April 8, 2008 Comment #1

In regard to immunotoxicity outcomes, the res ponses to comments are, with one exception, satisfactory. The exception is th e lack of a discussion of the reported immunotox icity results. No context is presented for interpreting the results. It would be helpful to include estimated blood alcohol levels (BAL) to put the reported suppression in context. For exampl e, does exposure to 20,000mg EtOH/m 3 cause extreme intoxication? The re port should provide a discussion of the biological effects, the doses that caused the effects and the likely human effects at similar BALs.

RG Response to EPA April 8, 2008 Comment 1:

EPA comments to draft 1 of this report [June 9, 2003] were similar to thos e submitted with draft 2 requesting BAL levels either meas ured or estimated and a discussi on of biological mechanisms. The RG response to these original comments [s ee reviewer checklist su bmitted with draft 2, Nov 12, 2007] indicated that BAL levels were not measur ed and that an extensive discussion of possible mechanisms was beyond the scope of this study. The stud y was performed as pa rt of a series of plaque-forming assays on baseline gasoline [BG] and 6 gasoline/oxygenate blends to identify and compare immunotoxic potential of these materials. It was not designed as a definitive assay for BG+ETOH immunotoxicity. A discussion such as that recommended by the EPA reviewers in the absence of measured BAL data is speculative a nd beyond the scope of this study, a consideration acknowledged by the initial EPA reviewer. Dr. Kimber White, the investigator on this study appreciated the initial EPA reviewer’s comments but considered it more appropriate to address these issues in a peer-reviewed publication of the immunotoxicity results of all seven materials rather than in this single study. The RG continues to concur with this position. Also, the RG points

out that the test material co ntained approximately 10% ethanol and 90% gasoline vapor. The exposure to ethanol from this mixt ure would never reach 20,000mg EtOH/m 3

.

Appendix Y: In Vivo-In Vitro Rat Peripheral Bl ood Lymphocyte Sister Chromatid Exchange Assay (SCE). On page 12, the report inco rrectly states that in the st atistical analyses of male rat lymphocytes there was a statis tically significant increase in SCE frequency at 2000mg/m 3 . An examination of the data in the table shows that this was instead a statistically significant decrease. In addition, according to the report, a one-tailed test was used. A one -tailed test is appropriate for detection of increases only. C onsequently, there is an inconsistency between the method and the reported results that needs to be resolved.

RG Response to EPA April 8, 2008 Comment 2:2 The RG appreciates being notified of this error and the text wi ll be corrected. The RG also acknowledges the appropriate application of one-t ailed tests. The purpose of performing an SCE assay was to determine whether the test material wa s able to induce perturbation of genetic material as evidence by an increase in SCE in peripheral blood lymphocy tes and a one-tailed test was appropriate for this purpose. The results of this assay were negative and the EPA reviewer of the

first draft considered the data evaluation adequate to support a c onclusion of “no effect”. No

revisions of the statistics methods will be made.

EPA April 8, 2008 Comment #3

Appendix X: Micronucleus assay. The discussion of the results of the micronucleus (MN) assay is problematic. A small but statistically significant in crease in the MN frequency is reported in the polychromatic (immature) erythocytes (PCE). The re port states that this fi nding was biologically irrelevant due to the fact that the negativ e control was abnormally low, thus enhancing the statistical significance of the highest concentration examined. The report states that is the statistically significant high dose is removed there were no significant differences when the 2 lower doses were compared to the control. However , we disagree with this approach and the resulting

conclusion.

By examining the data presented in the Appendix, it is apparent that this study, for reasons unknown, was relatively inse nsitive for detectin g MN induction. An examina tion of the historical positive controls with cyclophosphamide (assumed to be 40mg/kg) showed a mean frequency of 48 to 49 micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes examined (MN PCE/2000). In the present study, where th e authors dismiss the positive findings for ethanol due to due to abnormally low negative control ( 0.0 and 0.2 for males and females respectively), the cyclophosphamide positive controls range only betw een 14 and 8 MN PCEs/2000 for the male and female immature erythrocytes, resp ectively. This is roughly 6- to 4-fold lower than their usual historical positive control. In addition, the histor ical negative control data are shown to be between 0.71 and 0.94MN PCEs/2000. Thus, the negati ve controls for the curre nt ethanol study are approximately 8-fold lower than normal. Since the historical positive were very low dismissing the positive ethanol findings due to a lo w negative control is misleading. This suggests th at overall the study had low sensitivity for dete cting MN, and that the findings for ethanol ma y in fact be higher and more significant than indicated if the study were run with its usual sens itivity for detecting MN.

Therefore, it is not valid at this time to dism iss the positive findings for MN induction by ethanol, and a definitive results (eith er positive or negative) was not conclusively shown.

RG Response to EPA April 8, 2008 Comment 3:

The RG acknowledges the concerns raised by the EPA reviewer of draft 2 of this study. The study was reviewed carefully by the RG for the very reasons cited above. Our conclusion to submit the study to EPA as final was determined as follows.

• The ratio of immature to mature erythrocytes did not indicate any test material induced cell toxicity.3 • The low average values for negative control anim als resulted from the absence of MN-PCE in any of the 5 male animals and 4/5 female rats [ one female with MN-PCE]. The historical data for this laboratory gave an average of 0.71 fo r individuals and 0.94 for group means. However, the distribution figures indicated that for individual occurrenc es, 50% cells had no micronuclei and approximately 22% contained 0-0.2 occurren ces for group averages. In addition, group occurrences for negative control values demonstrated 23% 0.6- 0.8 micronuclei, suggesting that the incidence seen at BG+ETOH doses could have been spontaneous.

• The incidence of micronucleated polychromatic erythrocytes in the negative control or BG+ETOH treated groups has no more than 1- 2 MN-PCE per 2000 cells examined with the exception of one 20,000mg/m 3 rat with 3. In contrast, the cyclophosphamide positive control, although the overall average was lower than the historical positive control mean, had MN-PCE in every animal examined with an inciden ce range of 5-22 MN-PCE in 2000 cells examined.

• The study was performed using rats exposed for 28 days in whole body inhalation chambers as part of a 90-day study, which had been termin ated by the time these data were submitted.

• Results from the micronucleus assay performed w ith unleaded baseline gasoline alone indicated that baseline gasoline did not induce increased incidence of MN-PCE. This study has already been reviewed and accepted by EPA.

• Review of the published literature on the genetic toxicity of ethanol indicated that overall ethanol is not considered mutage n or clastogenic and has sometimes been used a solvent control. (see review by Phillips, BJ and Jenkinson, P. 2001. Mutagenesis 16(2): 91-101).

Review of all this information suggested that, despite the low va lues reported for negative and positive controls in this study, BG+ETOH condensate is unlikely to induce biologically significant increases in micronucleated polychrom atic erythrocytes or to be cons idered clastogenic. Both the scientific peer reviewers and the EPA reviewer of draft 1 apparently considered this conclusion to be adequate as well. The EPA review of the fi rst draft (June 9, 2003) stated “The micronucleus assay and the SCE assay appear to have been conducted according to re cognized and appropriate protocols. The conclusions of no effects of ga s + ETOH are supported by the data. The reviewers’ minor comments have been adequately addressed”. This is the entirety of their comments on the genetic toxicity studies. Therefore th e study is submitted to EPA as final.